Professional Documents
Culture Documents
10433
New Haplotypes in ATP Synthase Subunit 6 and Acute Lymphoblastic Leukemia
RESEARCH ARTICLE
New Haplotypes of the ATP Synthase Subunit 6 Gene of
Mitochondrial DNA are Associated with Acute Lymphoblastic
Leukemia in Saudi Arabia
Haitham Ahmed Yacoub1,2*, Wael Mahmoud Mahmoud3,4, Hatim Alaa-Eldeen
El-Din El-Baz5,6, Ola Mohamed Eid3, Refaat Ibrahim El-Fayoumi7,8, Maged
Mostafa Mahmoud9,10*, Steve Harakeh11 , Osama HA Abuzinadah2
Abstract
Background: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and
represents approximately 25% of cancer diagnoses among those younger than 15 years of age. Aim and Objectives:
This study investigated substitutions in the ATP synthase subunit 6 gene of mitochondrial DNA (mtDNA) as
a potential diagnostic biomarker for early detection and diagnosis of acute lymphoblastic leukemia. Based
on mtDNA from 23 subjects diagnosed with acute lymphoblastic leukemia, approximately 465 bp of the ATP
synthase subunit 6 gene were amplified and sequenced. Results: The sequencing revealed thirty-one mutations
at 14 locations in ATP synthase subunit 6 of mtDNA in the ALL subjects. All were identified as single nucleotide
polymorphisms (SNPs) with a homoplasmic pattern. The mutations were distributed between males and females.
Novel haplotypes were identified in this investigation: haplotype (G) was recorded in 34% in diagnosed subjects;
the second haplotype was (C) with frequency of 13% in ALL subjects. Neither of these were observed in control
samples. Conclusions: These haplotypes were identified for the first time in acute lymphoblastic leukemia patients.
Five mutations able to change amino acid synthesis for the ATP synthase subunit 6 were associated with acute
lymphoblastic leukemia. This investigation could be used to provide an overview of incidence frequency of acute
lyphoblastic leukemia (ALL) in Saudi patients based on molecular events.
Keywords: Acute lymphoblastic leukemia - ATP synthase subunit 6 gene - mtDNA - biomarker
Asian Pac J Cancer Prev, 15 (23s), 10433-10438
Introduction
Acute lymphoblastic leukemia (ALL) is the most
common cancer diagnosed in children and represents
approximately 25% of cancer diagnoses among children
younger than 15 years of age (National Cancer Institute,
2012 a; b). ALL occurs at an annual rate of 35 to 40
cases per 1 million people in the United States (Smith et
al., 1999; National Cancer Institute, 2012a; b). Among
children and adolescents younger than 20 years of age,
2,900 are diagnosed with ALL each year in the United
States (Smith et al., 1999; Dores et al., 2012). Over the
past 25 years, there has been a gradual increase in the
incidence of ALL (Shah and Coleman, 2007; National
Cancer Institute, 2012 a; b).
A sharp peak in the occurrence of ALL is observed
among children of 2 to 3 years of age (more than 90
Cell Biology Department, 6Biochemistry Department, Genetic Engineering and Biotechnology Division, 10Molecular Genetics
and Enzymology Department, 3Human Cytogenetics, Human Genetics & Genome Research Division, National Research Center,
Cairo, 8Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt, 2Biological Sciences Department, Faculty
of Sciences, 4Department of Medical Genetics, 5Clinical Biochemistry Department, Faculty of Medicine - North Jeddah Branch,
7
Medical Laboratories Technology Department, Faculty of Applied Medical Sciences, 9King Fahd Medical Research Center, King
Abdulaziz University, 11Special Infectious Agents Unit, Jeddah, Kingdom of Saudi Arabia *For correspondence: haithamyacoub46@
gmail.com, magedmostafa27@gmail.com
1
10433
Fragment name
CTGTTCGCTTCATTCATTGCC
GTGGCGCTTCCAATTAGGTG
10434
Concurrent chemoradiation
Radiotherapy
Chemotherapy
Remission
Persistence or recurrence
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.23.10433
New Haplotypes in ATP Synthase Subunit 6 and Acute Lymphoblastic Leukemia
Control.1
GCAGTACTGATCATTCTATTTCCCCCTCTATTGATCCCCACCTCCAAATATCTCATCAAC 60
19
GCAGTACTGATCATTCTATCTCCCCCTCTATTGATCCCCACCTCCAAATATCTCATCAAC 60
Control.4
GCAGTACTGATCATTCTATTTCCCCCTCTATTGATCCCCACCTCCAAATATCTCATCAAC 60
11
GCAGTACTGATCATTCTATTTCCCCCTCTATTGACCCCCACCTCCAAATATCTCATCAAC 60
2
GCAGTACTGATCATTCTATTTCCCCCTCTATTGACCCCCACCTCCAAATATCTCATCAAC 60
Control.1
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAACC 120
5
AACCGACTAATCACCGCCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAACC 120
Control.3
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAACC 120
12
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAATCTCAAAACAAATGATAACC 120
15
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAATCTCAAAACAAATGATAACC 120
Control.4
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAATCTCAAAACAAATGATAACC 120
3
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
7
AACCGACTAATTACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
4
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
20
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
10
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
11
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
18
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
2
AACCGACTAATCACCACCCAACAATGACTAATCAAACTAACCTCAAAACAAATGATAGCC 120
Control.4
GCCACAACTAACCTCCTCGGACTCCTGCCTCACTCATTTACACCAACCACCCAACTATCT 240
20
GCCACAACTAACCTCCTCGGACTCCTGCCCCACTCATTTACACCAACCACCCAACTATCT 240
Control.1
8
ATAAACCTAGCCATGGCCATCCCCTTATGAGCGGGCGCAGTGATTATAGGCTTTCGCTCT 300
ATAAACCTAGCCATGGCCATCCCCCTATGAGCGGGCGCAGTGATTATAGGCTTTCGCTCT 300
Control.4
1
ATACTAGTTATTATCGAAACCATCAGCCTACTCATTCAACCAATAGCCCTGGCCGTACGC 420
ATACTAGTTATTATCGAAACCATCAGCCTACTCATTCAACCAATAGCCCGGGCCGTACGC 420
Control.4
CTAACCGCTAACATTACTGCAGG--CCCCCTACTCATGCACCTAAT 464
18
CTAACCGCTAACATTAGTGCAGG--CCCCCTACTCATGCACCTAAT 464
2
CTAACCGCTAACATTACTGGGGGGCCCCCTACTCATGCACCTAAT 465
1
CTAACCGATAACATTGGTGGGGGGCCCCCTACTCATGCACCTAAT 465
Figure.1. Clastal W 2.1 Multiple Sequence Alignment among Acute Lymphoblastic Leukemia and Control
Subjects. Changes are marked bold, underlined in red
Thirty-one mutant sites were exploited at fourteen
location in ATP synthase subunit 6 of ALL subjects
as shown in (Table 2). All variation in ATP synthase
subunit 6 of mtDNA were identified as single nucleotide
polymorphisms (SNPs) with homoplasmic pattern. The
excited mutations were distributed in between male and
female.
Allele frequencies in ATP synthase subunit 6 of mtDNA
in acute lymphoblastic leukemia patients
The normal and mutant allele frequencies of ATP
synthase subunit 6 of mtDNA in ALL patients were shown
in (Table.2). The results show that fourteen mutant site
at ATP synthase subunit 6 gene were observed in this
investigation. The results showed that the mutant allele
at ATP synthase subunit 6 of mtDNA in ALL subjects had
different frequency up to 34% and most of mutants alleles
have not been conducted before in any other studies.
Novel haplotype in acute lymphoblastic leukemia patients
Novel haplotypes were identified in ATP synthase
subunit 6 gene of mtDNA in ALL subjects in this
investigation as shown in Figure 1. Haplotype (G) was
recorded 34% in diagnosed patients and it was not
shown in control ones. The second haplotype was (C)
with frequency of 13% in ALL subjects and it was not
observed in control samples as well. These haplotypes
were identified at the first time in acute lymphoblastic
leukemia patients and have not been conducted before in
Normal allele
T
0.96
T 0.92
A 0.96
C 0.95
C 0.92
A 0.66
T 0.96
T 0.96
T 0.96
C 0.96
G 0.96
- 0.92
A 0.87
A 0.96
C 0.92
Mutant allele
C
0.04
C 0.08
G 0.04
T 0.05
T 0.08
G 0.34
C 0.04
C 0.04
G 0.04
G 0.04
A 0.04
G 0.08
C 0.13
G 0.04
G 0.08
10435
Methionine to Isoleucine
Alanine to Aspartic Acid
Threonine to Glycine
Histidine to proline
Alanine to Glycine
0.34
0.04
0.04
0.13
0.08
10436
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.23.10433
New Haplotypes in ATP Synthase Subunit 6 and Acute Lymphoblastic Leukemia
November 2012 SEER data submission, posted to the SEER
in different regions within a certain genome and most of
web site, April 2013, Section 28. also available online. Last
these mutations were reported as homoplastic in nature
accessed April 04, 2014.
(Chatterjee et al., 2006). In addition, it illustrates how
Childhood cancer by the ICCC. In: Howlader N, Noone AM,
mtDNA alterations activate mitochondria-to-nucleus
Krapcho M, et al., eds.: SEER Cancer Statistics Review,
retrograde signaling to modulate the expression of relevant
1975-2010. Bethesda, Md: National Cancer Institute, based
nuclear genes or induce epigenetic changes and promote
on November 2012 SEER data submission, posted to the
malignant phenotypes in cancer cells.
SEER web site, April 2013, Section 29. Also available
In this investigation, we were used direct DNA
online. Last accessed June 26, 2014.
sequencing method for ATP synthase subunit 6 gene
Coller HA, Khrapko K, Bodyak ND, et al (2001). High frequency
of homoplasmic mitochondrial DNA mutations in human
of mtDNA which has advantage when compared with
tumors can be explained without selection. Nat Genet, 28,
alternative techniques for identification such as PCR147-50.
RFLP, PCR using specific primers offers the advantages of
Cook CC, Higuchi M. (2011). The awakening of an advanced
being less expensive and more useful for routine analysis
malignant cancer: An insult to the mitochondrial genome.
of large numbers of samples.
Biochim Biophys Acta, 1820, 652-62.
In conclusion, in our study, thirty-one mutant sites
DiMauro S, Schon EA. (2001). Mitochondrial DNA mutations
were exploited at 14 location in ATP synthase subunit 6
in human disease. Am J Med Genet, 106, 18-26.
of mtDNA in ALL subjects and these kind of mutations
Dores GM, Devesa SS, Curtis RE, Linet MS, Morton LM.
have not been conducted before in any other studies.
(2012). Acute leukemia incidence and patient survival
among children and adults in the United States, 2001-2007.
Novel haplotypes were identified in ATP synthase subunit
Blood, 119, 34-43.
6 gene of mtDNA in this investigation. Haplotype (G)
Fliss MS, Usadel H, Caballero OL, et al (2000). Facile detection
was recorded 34% in diagnosed patients and it was not
of mitochondrial DNA mutations in tumors and bodily fluids.
shown in control ones. The second haplotype was (C)
Science, 287, 2017-9.
with frequency of 13% in ALL subjects and it was not
Higuchi M (2012). Roles of mitochondrial DNA changes on
observed in control samples as well. These haplotypes
cancer initiation and progression. Cell Biol Res Ther, 1, 2-4.
were identified at the first time in acute lymphoblastic
Levin BC, Cheng H, Reeder DJ (1999). A human mitochondrial
leukemia patients and have not been conducted before in
DNA standard reference material for quality control in
any other studies. Five mutations were be able to change
forensic identification, medical diagnosis and mutation
detection. Genomics, 55, 135-46.
amino acids synthesis at ATP synthase subunit 6 and are
Lu J, Sharma LK, Bai Y (2009). Implications of mitochondrial
associated with acute lymphoblastic leukemia patients.
DNA mutations and mitochondrial dysfunction in
This investigation could be used to provide an overview
tumorigenesis. Cell Res, 19, 802-15.
of incidence frequency of acute lyphoblastic leukemia
Parrella P, Seripa D, Matera MG, et al (2003). Mutations of
(ALL) in Saudi patients based on molecular level.
the D310 mitochondrial mononucleotide repeat in primary
tumors and cytological specimens. Cancer Lett, 190, 73-7.
Acknowledgements
Sabir N, Iqbal Z, Aleem A, et al (2012). Prognostically significant
fusion oncogenes in Pakistani patients with adult acute
We gratefully thank the Center of Excellence
lymphoblastic leukemia andtheir association with disease
biology and outcome. Asian Pac J Cancer Prev, 13, 3349-55.
in Genomic Medicine Research (CEGMR) at King
Shaikh MS, Ali SS, Khurshid M, Fadoo Z (2014). Chromosomal
Abdulaziz University, Kingdom of Saudi Arabia for
abnormalities in Pakistani children with acute lymphoblastic
providing us with DNA samples of ALL patients through
leukemia. Asian Pac J Cancer Prev, 15, 3907-9.
the communications with Dr. Refaat Elfayoumi. The
Smith MA, Gloeckler-Ries LA, Gurney JG, Ross JA (1999).
authors report no conflicts of interest. The authors alone
Leukemia. in Ries LAG, Smith MA, Gurney JG ,et al
are responsible for the content and writing of this article.
(Eds). Cancer Incidence and Survival among Children and
Adolescents: United States SEER Program 1975-1995
(pp.17-34). Bethesda, Md: National Cancer Institute. SEER
References
program: NH, Pub N. 99-4649.
Awan T, Iqbal Z, Aleem A, et al (2012). Five most common
Wallace DC (1992). Diseases of the mitochondrial DNA. Annu
prognostically important fusion oncogenes are detected
Rev Biochem, 61, 1175-212.
in the majority of Pakistani pediatricacute lymphoblastic
Yacoub HA, Mahmoud WM, El-Baz HA, et al (2014). Novel
leukemia patients and are strongly associated with disease
mutations in the displacement loop of mitochondrial dna
biology and treatment outcome. Asian Pac J Cancer Prev,
are associated with acute lymphoblastic leukemia: a genetic
13, 5469-75.
sequencing study. Asian Pac J Cancer Prev, 15, 9283-9.
Carew JS, Zhou Y, Albitar M, et al (2003). Mitochondrial DNA
Yu M (2012) Somatic mitochondrial DNA mutations in human
mutations in primary leukemia cells after chemotherapy:
cancers. Adv Clin Chem, 57, 99-138.
clinical signicance and therapeutic implications. Leukemia,
Zastawny TH, Dabrowska M, Jaskolski T, et al (1998).
17, 1437-47.
Comparison of oxidative base damage in mitochondrial and
Carew JS, Huang P. (2002). Mitochondrial defects in cancer.
nuclear DNA. Free Rad Biol Med, 24, 722-5.
Mol Cancer, 9,1-9.
Chatterjee A, Mambo E, Sidransky D (2006). Mitochondrial
DNA mutations in human cancer. Oncogene , 25, 4663-74.
Childhood cancer. In: Howlader N, Noone AM, Krapcho
M, et al., eds.: SEER Cancer Statistics Review, 19752010. Bethesda, Md: National Cancer Institute, based on
Asian Pacific Journal of Cancer Prevention, Vol 15, 2014
10437
10438